Telomeres are crucial for maintaining the integrity of chromosomes, and telomeric dysfunction has emerged as a cause for cell senescence and possible stem cell loss of function, leading to impaired tissue function and regeneration. Telomere dysfunction has been implicated in diverse age-associated diseases, such as cancer, atherosclerosis and osteoporosis. The segmental progeroid disease Werner Syndrome is characterized by the stochastic loss of telomeres and premature cell senescence, and is caused by loss of the WRN helicase/exonuclease protein. WRN protein interacts with telomeric proteins POT1and TRF2, which are both implicated in the processing of alternate structures at telomeric ends, including G-quadruplexes (G4 DNA), t-loop/d-loops, and Holliday Junctions (HJ). Such structures are preferred substrates for WRN protein, and improper processing of these structures at telomeres can lead to telomere loss and genomic instability. We hypothesize that alternate structures of DNA exist in dynamic equilibrium in telomeric DNA and the equilibrium is modulated by WRN in cooperation with telomere-binding proteins, which we will test using several biophysical and biochemical methods. In specific aim 1, we will test whether WRN helicase/exonuclease and POT1 cooperate in resolving telomeric G4 DNA, an impediment to DNA polymerase. G4 DNA formation and dissociation will be tracked using single molecule fluorescence resonance energy transfer (FRET). In specific aim 2, we will use Atomic Force Microscopy and computational modeling to determine the arrangement and equilibrium of G4 DNA structures on long telomeric DNA molecules in which multiple states are possible. We will then study whether POT1 binding can cooperatively destabilize G4 DNA on longer molecules. In specific aim 3, we will determine the ability of TRF2 and POT1 to modulate WRN activity in dissociating HJ structures. The HJ constructs will be composed of two telomeric and two unique-sequence DNA arms, with each arm individually labeled with a fluorophore. Our unique fluorophore-labeling system will permit us to compare the fates of each telomeric arm relative to the constructs mixed-sequence arms in strand displacement gel assays. PUBLIC HEALTH RELEVANCE: Improper processing of telomeric DNA structures may underlie several aging-related diseases. This proposal analyzes the mechanisms for telomeric DNA processing, which should provide insights into the aging process and may assist in finding therapeutic targets to arrest or delay genomic instability and telomere loss.